Pyruvate Dehydrogenase Variants, A Microorganism Comprising The Same And A Method For Producing L-amino Acid Using The Same

Abstract

The present disclosure relates to a novel pyruvate dehydrogenase variant, a polynucleotide encoding the pyruvate dehydrogenase variant, a microorganism of the genus Corynebacterium producing L-amino acid, which includes the pyruvate dehydrogenase variant, and a method for producing an L-amino acid using the microorganism.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation application of U.S. application Ser. No. 15/557,013 filed Sep. 8, 2017, now pending, which is a U.S. national phase application of PCT/KR2016/002594, filed Mar. 15, 2016, which claims priority to KR Application No. 10-2015-0037654, filed Mar. 18, 2015. U.S. application Ser. No. 15/557,013 is herein incorporated by reference in its entity.

STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200187_431C1_SEQUENCE_LISTING.txt. The text file is 155 KB, was created on May 29, 2019, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

[0003] The present disclosure relates to a novel pyruvate dehydrogenase variant, a polynucleotide encoding the pyruvate dehydrogenase variant, a microorganism of the genus Corynebacterium, having an ability to produce L-amino acid which comprises the variant, and a method for producing an L-amino acid using the microorganism.

BACKGROUND ART

[0004] Pyruvate dehydrogenase multienzyme complex (PDHC) is an enzyme converting pyruvate generated during glycolysis into acetyl-CoA and is an important enzyme for determining carbon introduction to TCA cycle. PDHC consists of pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p), and dihydrolipoamide dehydrogenase (E3p). Among them, E1p enzyme is encoded by aceE gene. Although the changes in L-lysine production in strain producing L-lysine due to deleting and weakening of aceE gene (Blombach et al., Appl. Microbiol. Biotechnol., 76: 615, 2007; Buchholz J et al., Appl Environ Microbiol., 79 (18): 5566-75, 2013) have been known, there has been no report regarding the E1p variant, which can improve abilities to produce L-amino acid.

DISCLOSURE

Technical Problem

[0005] The present inventors have made extensive efforts to develop a E1p variant that can be used in the production of an L-amino acid at high concentration and a microorganism using the same, and as a result, have developed the E1p variant and discovered that the L-amino acid can be produced in high yield from the microorganism containing the E1p variant, thereby completing the present disclosure.

Technical Solution

[0006] An object of the present disclosure is to provide a novel pyruvate dehydrogenase variant.

[0007] Another object of the present disclosure is to provide a polynucleotide encoding the pyruvate dehydrogenase variant.

[0008] A further object of the present disclosure is to provide a microorganism of the genus Corynebacterium producing L-amino acid, which includes the pyruvate dehydrogenase variant.

[0009] A still further object of the present disclosure is to provide a method for producing an L-amino acid, including: (a) culturing the microorganism of the genus Corynebacterium producing L-amino acid, which includes the pyruvate dehydrogenase variant, in a medium to produce an L-amino acid; and (b) recovering the L-amino acid from the cultured microorganism or the medium.

Advantageous Effects of the Present Invention

[0010] An enzyme with a weakened activity can be obtained using the pyruvate dehydrogenase variant of the present disclosure. As such, an L-amino acid can be produced with high efficiency using the microorganism, which includes the pyruvate dehydrogenase variant with weakened activity, compared to an L-amino acid-producing microorganism, which includes the wild type pyruvate dehydrogenase protein. Additionally, the microorganism of the present disclosure enables an effective production of an L-amino acid while rarely inhibiting the growth of the microorganism, unlike a pyruvate dehydrogenase-deleted microorganism. For example, lysine, as an essential amino acid for animal feed, is required to be produced in a large-scale from the industrial aspect. Therefore, the production of L-lysine with high efficiency as in the present disclosure can reduce the cost for manufacturing animal feed.

BEST MODE

[0011] An aspect of the present disclosure provides a pyruvate dehydrogenase variant which includes at least one amino acid substitutions in a region of amino acids at positions from 190 to 205 or in a region of amino acids at positions from 415 to 440 of SEQ ID NO: 1.

[0012] As used herein, the term "pyruvate dehydrogenase" refers to one of the enzymes constituting a pyruvate dehydrogenase multienzyme complex (PDHC), which is involved in the conversion of pyruvate into acetyl-CoA. As used herein, pyruvate dehydrogenase is not particularly limited as long as it has the corresponding activity, and it may be a pyruvate dehydrogenase derived from a microorganism of the genus Corynebacterium, specifically, Corynebacterium glutamicum, but is not limited thereto. For example, the pyruvate dehydrogenase may be an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a homology of at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher to the amino acid sequence of SEQ ID NO: 1. The E1p protein having the amino acid sequence of SEQ ID NO: 1 may be encoded by aceE gene having the polynucleotide sequence of SEQ ID NO: 2, but is not limited thereto. Additionally, if an amino acid sequence has a homology to the above sequence and has substantially the same or corresponding biological activity to the protein of SEQ ID NO: 1, it is obvious that the amino acid sequence with a deletion, a modification, a substitution, or an addition should also belong to the scope of the present disclosure. In the present disclosure, any polynucleotide sequence encoding pyruvate dehydrogenase may belong to the scope of the present disclosure. For example, the polynucleotide sequence may be a polynucleotide sequence which has a homology of at least 75%, specifically at least 80%, more specifically 85%, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, to the amino acid sequence of SEQ ID NO: 2. Additionally, based on the codon degeneracy or considering the codons preferred by organisms to express the protein, the polynucleotide sequence encoding the protein may have various variants on the coding region within the scope not changing the amino acid sequence of the protein being expressed from the coding region.

[0013] The pyruvate dehydrogenase variant according to the present disclosure may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 amino acid substitutions in the region of amino acids at positions from 190 to 205 or from 415 to 440 of SEQ ID NO: 1.

[0014] In particular, the pyruvate dehydrogenase variant according to the present disclosure may include at least one, at least two, at least three, or at least four amino acid substitutions in the region of amino acids at positions from 190 to 205 of SEQ ID NO: 1. Specifically, the amino acid substitution in the region of amino acids at positions from 190 to 205 of SEQ ID NO: 1 may be selected from the group of amino acids at positions 190, 195, 199, and 201, but is not limited thereto.

[0015] The substitution in the region of amino acids at positions from 190 to 205 of SEQ ID NO: 1 may be a substitution of at least one amino acid in the region of amino acids at positions from 190 to 205 with a different kind of an amino acid, more specifically, a substitution of at least one amino acid at positions 190, 195, 199, and 201 with a different kind of an amino acid, and even more specifically, at least one selected from the group consisting of a substitution of glutamic acid at position 190 with valine (E190V), a substitution of glutamine at position 195 with histidine (Q195H), a substitution of proline at position 199 with serine (P199S), and a substitution of tyrosine at position 201 with alanine (Y201A), but is not particularly limited thereto.

[0016] Additionally, the pyruvate dehydrogenase variant according to the present disclosure may include at least one, at least two, at least three, at least four, at least five, or at least six amino acid substitutions in the region of amino acids at positions from 415 to 440 of SEQ ID NO: 1. Specifically, the amino acid substitution in the region of amino acids at positions from 415 to 440 of SEQ ID NO: 1 may be selected from the group consisting of amino acids at positions 418, 428, 432, 435, and 438.

[0017] The substitution in the region of amino acids at positions from 415 to 440 of SEQ ID NO: 1 may be a substitution of at least one amino acid in the region of amino acids at positions from 415 to 440 with a different kind of an amino acid, specifically, a substitution of at least one amino acid at positions 418, 428, 432, 435, and 438 with a different kind of an amino acid, and even more specifically, at least one selected from the group consisting of a substitution of tyrosine at position 418 with histidine (Y418H), a substitution of asparagine at position 428 with alanine (N428A), a substitution of glutamine at position 432 with glutamic acid (Q432E), a substitution of glutamine at position 432 with alanine (Q432A), a substitution of lysine at position 435 with alanine (K435A), and a substitution of leucine with proline (L438P), but is not particularly limited thereto.

[0018] More specifically, the pyruvate dehydrogenase variant may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 14 to 33.

[0019] The pyruvate dehydrogenase variant may include not only the proteins having the amino acid sequences of SEQ ID NOS: 14 to 33 but also the pyruvate dehydrogenase variant having a homology of 75% or higher, specifically 80% or higher, more specifically 85% or higher, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher, to the amino acid sequences of SEQ ID NOS: 14 to 33, without limitation, as long as their pyruvate dehydrogenase activity is substantially weakened compared to that of the wild type. It is obvious that the amino acid sequences having substantially the same or corresponding biological activity with the proteins having the amino acid sequences of SEQ ID NOS: 14 to 33 should also belong to the scope of the present disclosure, although the amino acid sequences may have a deletion, a modification, a substitution, or an addition, in part of the sequences.

[0020] As used herein, the term "homology" refers to a percentage of identity between two polynucleotide or polypeptide moieties. The homology between sequences from a moiety to another moiety may be determined by the technology known in the art. For example, the homology may be determined by directly arranging the sequence information of two polynucleotide molecules or two polypeptide molecules using an easily accessible computer program. Examples of the computer program may include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign.TM. (DNASTAR Inc.), etc. Additionally, the homology between polynucleotides may be determined by hybridizing polynucleotides under the condition of forming a stable double-strand between the homologous regions, disassembling with a single strand-specific nuclease, followed by size determination of the disassembled fragments.

[0021] An aspect of the present disclosure provides a polynucleotide encoding the pyruvate dehydrogenase variant.

[0022] The pyruvate dehydrogenase variant is the same as explained above. Specifically, the polynucleotide encoding the pyruvate dehydrogenase variant may belong to the scope of the present disclosure as long as the polynucleotide encodes proteins having the amino acid sequences of SEQ ID NOS: 14 to 33. Furthermore, the polynucleotide may be any polynucleotide having a homology of 75% or higher, specifically 80% or higher, more specifically 85% or higher, and even more specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher, to the above polynucleotide sequence.

[0023] As used herein, the term "polynucleotide" refers to a polymer of nucleotides lengthwise chain-extended by a covalent bond of nucleotide units, in general DNA or RNA strand with a certain length, and in the present disclosure, it refers to a polynucleotide encoding the pyruvate dehydrogenase variant. The polynucleotide may have various nucleotide sequences encoding the same amino acid based on codon degeneracy. Additionally, for the optimization of the expression according to the type of host cells, the polynucleotide may have a codon-optimized sequence.

[0024] Still another aspect of the present disclosure provides a microorganism of the genus Corynebacterium producing L-amino acid, which includes the pyruvate dehydrogenase variant.

[0025] Specifically, the microorganism may include the pyruvate dehydrogenase variant by a mutation, or may be one transformed by a vector including a polynucleotide, which encodes the pyruvate dehydrogenase variant.

[0026] As used herein, the term "vector" refers to any carrier for cloning and/or transferring bases to a host cell. A vector may be a replicon to allow for the replication of other DNA fragment(s) which combined with the vector. "Replicon" refers to any genetic unit acting as the unit of DNA replication in vivo, that is, genetic units (e.g., plasmids, phages, cosmids, chromosomes, and viruses) replicable by self-regulation. As used herein, the term "vector" is not particularly limited as long as it can replicate in a host, and any vector known in the art may be used. The vector used in the construction of the recombinant vector may be in a natural state or plasmids, cosmids, viruses, and bacteriophages in a recombinant state. For example, as a phage vector or cosmid vector, pWE15, M13, .lamda.EMBL3, .lamda.EMBL4, .lamda.FIXII, .lamda.DASHII, .lamda.ZAPII, .lamda.gt10, .lamda.gt11, Charon4A, Charon21A, etc., may be used, and as a plasmid vector, those based on pDZ vector, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., may be used. The vectors that can be used in the present disclosure are not particularly limited but any known expression vector may be used. Specifically, pDZ (Korean Patent No. 10-0924065 is incorporated in its entirety as a reference in the present disclosure) may be used, but is not limited thereto.

[0027] As used herein, the term "transformation" refers to a process of introducing a gene into a host cell, thereby enabling the expression of the gene in the host cell. The transformed gene may include, without limitation, any gene whether it is inserted into the chromosome of a host cell and located therein or located outside the chromosome, as long as it can be expressed in the host cell. The polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct including all essential elements required for self-expression. The expression cassette may conventionally include a promoter operably connected to the gene, a transcription termination signal, a ribosome-binding domain, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a host cell as it is or in the form of polynucleotide construct and operably connected to a sequence necessary for its expression in the host cell, but is not limited thereto.

[0028] The microorganism may be any prokaryotic or eukaryotic microorganism as long as the microorganism includes the activity of the corresponding pyruvate dehydrogenase variant or the microorganism is transformed to express the corresponding protein. Examples of the microorganism may include microbial strains of the genus Escherichia, the genus Erwinia, the genus Serratia, the genus Providencia, the genus Enterobacteria, the genus Salmonella, the genus Streptomyces, the genus Pseudomonas, the genus Brevibacterium, the genus Corynebacterium, etc., specifically, a microorganism of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0029] Additionally, when the pyruvate dehydrogenase variant of the present disclosure is included in the microorganism having an ability to produce L-amino acid, the ability to produce L-amino acid can be significantly improved without considerably inhibiting the growth of cells, compared to the microorganism including the wild-type pyruvate dehydrogenase.

[0030] As used herein, the term "L-amino acid" refers to all L-amino acids that can be produced from various carbon sources through pyruvate, and specifically, may refer to an L-amino acid which does not go through with a process of converting pyruvate into acetyl CoA in a biosynthetic pathway. More specifically, the L-amino acid may include L-lysine, L-threonine, L-methionine, L-isoleucine, L-valine, L-leucine, or L-alanine, and even more specifically, L-lysine or L-valine.

[0031] The microorganism producing L-amino acid may include both eukaryotic and prokaryotic microorganisms that can produce L-amino acid in vivo, and examples of the microorganisms are the same as described above. The microorganism producing L-amino acid may be any microorganism without limitation as long as the microorganism has the ability to produce L-amino acid, including both wild-type strains and recombinant strains.

[0032] For example, a microorganism of the genus Corynebacterium producing L-lysine may be modified to be resistant to an L-lysine analog or have enhanced activity of the L-lysine biosynthesis-related protein compared to that of unmodified microorganism. Specifically, expression of at least one kind of gene related to L-lysine biosynthesis is improved by gene amplification; substitution or modification of sequences such as a promoter or a start codon; introduction of a modification for the improvement of expression, etc., but is not limited thereto.

[0033] Additionally, examples of the L-lysine biosynthesis-related gene may include the genes located on the L-lysine biosynthesis pathway, and specifically, dihydrodipicolinic acid synthasegene (dapA), asparto kinasegene (lysC), dihydrodipicolinic acid reductase gene (dapB), diaminopimelic acid decarboxylase gene (lysA), diaminopimelic acid dehydrogenase gene (ddh), phosphoenolpyruvate carboxykinase gene (ppc), aspartate semialdehyde dehydrogenase gene (asd), aspartase gene (aspB), and pyruvate carboxylase (Pyc), but is not limited thereto. Additionally, examples of the L-lysine biosynthesis-related gene may include transketolase (tkt), etc., present on the pentose phosphate pathway, but is not limited thereto.

[0034] In particular, the microorganism of the genus Corynebacterium producing L-lysine may exhibit the ability to produce L-lysine by including the modification, which is related to L-lysine production, disclosed in the art, but is not limited thereto.

[0035] The microorganism producing L-threonine may be a microorganism, which has a methionine requirement, a resistance to threonine analogs, a resistance to lysine analogs, a resistance to isoleucine analogs, and/or a resistance to methionine analogs, although not particularly limited thereto. Methionine analogs may be at least one compound selected from the group consisting of D,L-ethionine, norleucine, .alpha.-methylmethionine, and L-methionine-D,L-sulfoximine; threonine analogs may be at least one compound selected from the group consisting of .alpha.-amino-.beta.-hydroxy valeric acid and D,L-threonine hydroxamate; and lysine analogs may be at least one compound selected from the group consisting of S-(2-aminoethyl)-L-cysteine and .delta.-methyl-L-lysine.

[0036] Additionally, the microorganism producing L-threonine may include a microorganism, in which the activity of PckA involved in the conversion of oxaloacetate (OAA), which is an intermediate for L-threonine biosynthesis, into phosphoenolpyruvate (PEP) is weakened or inactivated; or a microorganism, in which the activity of TyrR, which inhibits lysC gene involved in the conversion of oxaloacetate into aspartate is weakened or inactivated; or a microorganism, in which the activity of GalR, which inhibits the expression of galP gene involved in the introduction of glucose is weakened or inactivated, but is not limited thereto.

[0037] The microorganism producing L-isoleucine may be a microorganism having a resistance to L-isoleucine or derivatives thereof, or a microorganism which was genetically manipulated to release the feedback inhibition by L-isoleucine or derivatives thereof. Examples of the derivatives of L-isoleucine may include 4-thiaisoleucine (thiaile) and isoleucine-hydroxamate (ileHx), but is not limited thereto.

[0038] The microorganism producing L-valine may be a microorganism having a resistance to L-valine or derivatives thereof, or a microorganism in which the enzyme in the biosynthesis pathway of L-valine was genetically manipulated to release the feedback inhibition by L-valine or derivatives thereof. Examples of the microorganism may include a microorganism including an acetohydroxy acid synthase variant, in which the feedback inhibition to L-valine is released, but is not limited thereto. Additionally, the microorganism may be one in which the expression of L-valine operon was modified to be enhanced, and for example, the microorganism may be one in which the expression of L-valine operon was enhanced due to the deletion of a part or entirety of the polynucleotide sequence encoding the leader peptide within the regulation region of L-valine operon (Korean Patent Application Publication No. 10-2014-0111421, the entirety of the specification of which may be incorporated as a reference but is not limited thereto).

[0039] In Still another aspect of the present disclosure provides a method for producing L-amino acid, including: (a) culturing the microorganism of the genus Corynebacterium producing L-amino acid, which includes the pyruvate dehydrogenase variant, in a medium to produce an L-amino acid; and (b) recovering the L-amino acid from the cultured microorganism or the medium.

[0040] The microorganism of the genus Corynebacterium producing L-amino acid, etc., are the same as described above.

[0041] As used herein, the term "culture" refers to growing the microorganism under appropriately and artificially controlled environmental conditions. The culture process of the present disclosure may be executed based on appropriate culture media and culture conditions known in the art. Specific conditions, such as culture temperature, culture time, pH value of culture medium, etc., may be determined by the general knowledge by one of ordinary skill in the art or the conventional method known in the art, and appropriately adjusted accordingly. Specifically, these known culture methods are described in references in details [Chmiel; Bioprozesstechnik 1. Einfuhrung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)]. Additionally, the culture methods may include batch culture, continuous culture, and fed-batch culture, and specifically, the culture may be performed continuously in a fed batch or repeated fed batch process, but are not limited thereto. The medium used in the culture must appropriately satisfy the requirements for specific strains. Examples of the carbon source to be used in the medium may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid, but are not limited thereto. These carbon sources may be used alone or in combination but are not limited thereto. Examples of the nitrogen sources to be used in the medium may include peptone, yeast extract, meat gravy, malt extract, corn steep liquor, soybean flour; and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources may also be used alone or in combination, but are not limited thereto. Examples of the phosphorus sources to be used in the medium may include dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts, etc., but are not limited thereto. Additionally, metal salts such as magnesium sulfate or iron sulfate required for growth may be contained in the medium. Lastly, essential materials for growth, such as amino acids and vitamins, may also be contained in addition to the materials described above. Additionally, precursors suitable for culture medium may be used. These sources may be added in an appropriate manner during the culture by a batch culture or a continuous culture to a culture, but the methods are not limited thereto.

[0042] Additionally, the pH value of a culture may be adjusted while culturing by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. During the culture period, an antifoaming agent, such as fatty acid polyglycol ester, may be added to prevent foam generation. Additionally, oxygen or an oxygen-containing gas may be injected into the culture in order to maintain an aerobic state of the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of a gas in order to maintain an anaerobic or microaerobic state of the culture. The culture temperature may generally be in the range from 27.degree. C. to 37.degree. C., and specifically, from 30.degree. C. to 35.degree. C. The culture may be continued until the desired amount of useful materials are obtained, and specifically for from 10 hours to 100 hours. L-amino acids may be released into the culture medium being cultured or may be contained in microorganisms.

[0043] Additionally, regarding the method of producing an L-amino acid of the present disclosure, the method of recovering the L-amino acid from a cultured microorganism or a culture is widely known in the art. The methods of recovering an L-amino acid may include filtration, anion exchange chromatography, crystallization, HPLC, etc., but are not limited thereto.

MODE FOR CARRYING OUT THE INVENTION

[0044] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the disclosure is not intended to be limited by these Examples.

Example 1: Construction of a Library of E1p Variants by Artificial Mutagenesis

[0045] In this Example, a vector library for the primary cross insertion within the chromosome was constructed in order to obtain E1p variants by the method described below. Error-prone PCR was performed for the Corynebacterium glutamicum ATCC13032-derived aceE gene (SEQ ID NO: 2), which encodes E1p (SEQ ID NO: 1), and modified aceE gene (2852 bp) randomly introduced with base substitution were obtained. Error-prone PCR was performed by the Genemorph II Random Mutagenesis Kit (Stratagene) using Corynebacterium glutamicum ATCC13032 genomic DNA as a template along with primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4).

TABLE-US-00001 primer 1 (SEQ ID NO: 3): 5'-TGGGA CCGGG AAACC GGG-3' primer 2 (SEQ ID NO: 4): 5'-GATTT ATCTG TCCCT TGA-3'

[0046] The amplified gene fragments were introduced with 0 to 3.5 base substitution per 1 kb, and PCR was performed for a total of 30 cycles under the following conditions: 96.degree. C. denaturation for 30 sec, annealing at 53.degree. C. for 30 sec, and polymerization at 72.degree. C. for 2 min.

[0047] The amplified fragments were linked to pCR2.1-TOPO vector (hereinafter, "pCR2.1") using the pCR2.1-TOPO TA cloning kit (Invitrogen), transformed into E. coli DH5.alpha., and plated on LB solid medium containing kanamycin (25 mg/L). The 20 different kinds of transformed colonies were selected and then plasmids were obtained therefrom. Upon analysis of the polynucleotide sequences of the plasmids, it was confirmed that modifications were introduced to mutually different positions with a frequency of 1.4 mutations/kb. About 20,000 transformed E. coli colonies were collected and their plasmids were extracted and named as "pCR2.1-aceE (mt)" library.

[0048] Additionally, a plasmid having the wild-type aceE gene to be used as the control strain was constructed. PCR was performed in the same manner as described above using the Corynebacterium glutamicum ATCC13032 genomic DNA as a template along with primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4). For the polymerase, PfuUltra High-Fidelity DNA polymerase (Stratagene) was used and the thus-constructed plasmid was named as "pCR2.1-aceE(WT)."

Example 2: Construction of an aceE-Deletion Strain

[0049] An aceE-deletion strain was constructed for the introduction of the pCR2.1-aceE (mt) library using the KCCM11016P (the above microorganism was first published as KFCC10881 and re-deposited to an international depositary authority under the Budapest Treaty and assigned the Accession No. KCCM11016P; Korean Patent No. 10-0159812) strain as a parent strain.

[0050] For the construction of an aceE-deletion vector, PCR was performed using the chromosome of the wild-type Corynebacterium glutamicum ATCC13032 as a template along with primer 3 (SEQ ID NO: 5), primer 4 (SEQ ID NO: 6), primer 5 (SEQ ID NO: 7), and primer 6 (SEQ ID NO: 8).

TABLE-US-00002 primer 3 (SEQ ID NO: 5): 5'-GCAGG TCGAC TCTAG ATGCG ATTCG CGTCA AACGT G-3' primer 4 (SEQ ID NO: 6): 5'-GTCCC TTGAG GTGAT GTGAA TCCAT CCACT-3' primer 5 (SEQ ID NO: 7): 5'-AGTGG ATGGA TTCAC ATCAC CTCAA GGGAC-3' primer 6 (SEQ ID NO: 8): 5'-CCGGG GATCC TCTAG ACGAA GCGCC GTGAG CAATT C-3'

[0051] PCR was performed under the following conditions: denaturation at 95.degree. C. for 5 min; 30 cycles of denaturation at 95.degree. C. for 30 sec, annealing at 55.degree. C. for 30 sec, and polymerization at 72.degree. C. for 30 sec; and polymerization at 72.degree. C. for 7 min.

[0052] As a result, SEQ ID NO: 9 (521 bp) and SEQ ID NO: 10 (538 bp), which respectively include the 5'terminus and 3'terminus, were obtained.

[0053] PCR was performed using the amplified sequences of SEQ ID NO: 9 and SEQ ID NO: 10 as templates along with primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 8).

[0054] PCR was performed under the following conditions: denaturation at 95.degree. C. for 5 min; 30 cycles of denaturation at 95.degree. C. for 30 sec, annealing at 55.degree. C. for 30 sec, and polymerization at 72.degree. C. for 60 sec; and polymerization at 72.degree. C. for 7 min.

[0055] As a result, SEQ ID NO: 11 with a size of 1029 bp (hereinafter, .DELTA.aceE), in which the 5' terminus and 3' terminus of the aceE gene were connected, was amplified.

[0056] The pDZ vector (Korean Patent No. 10-0924065), which cannot replicate in Corynebacterium glutamicum, and the .DELTA.aceE fragment were treated with the restriction enzyme Xba I and ligated using a DNA ligase, and cloned to obtain a plasmid. The plasmid was named aspDZ-.DELTA.aceE.

[0057] The pDZ-.DELTA.aceE was transformed into Corynebacterium glutamicum KCCM11016P, which is a L-lysine producing strain, by electric pulse (Appl. Microbiol. Biothcenol. (1999) 52: 541-545), and the transformed strain was obtained from a selective medium containing kanamycin (25 mg/L). A strain, in which the aceE gene was inactivated by .DELTA.aceE, the DNA fragment inserted into the genome via secondary cross-over, was obtained and named as KCCM11016P.DELTA.aceE.

Example 3: Construction of an E1p Artificial Variant Library and Screening of Strains with Enhanced Ability to Produce L-Lysine

[0058] The constructed pCR2.1-aceE (mt) library, was transformed into the KCCM11016P.DELTA.aceE strain as a parent strain by homologous recombination, plated on a complex plate medium containing kanamycin (25 mg/L), and about 10,000 colonies were obtained, and each colony was named as "KCCM11016P.DELTA.aceE/pCR2.1-aceE(mt)-1" to "KCCM11016P/pCR2.1-aceE(mt)-10000," respectively.

[0059] Additionally, the thus-constructed pCR2.1-aceE (WT) vector was transformed into KCCM11016P.DELTA.aceE to obtain a control strain, and named as "KCCM11016P.DELTA.aceE/pCR2.1-aceE (WT)."

[0060] <Complex Plate Medium (pH 7.0)>

[0061] Glucose 10 g, Peptone 10 g, Beef Extract 5 g, Yeast Extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitiol 91 g, and Agar 20 g (based on 1 L of distilled water)

[0062] The thus-obtained 10,000 colonies were respectively inoculated into a selective medium (300 .mu.L) and cultured in 96-deep well plates maintained at 32.degree. C. at a rate of 1000 rpm for about 24 hours. The amount of L-lysine production produced in the culture was analyzed by ninhydrin method (J. Biol. Chem. 1948. 176: 367-388). Upon completion of the culture, 10 .mu.L of the culture supernatant and 190 .mu.L of ninhydrin reaction solution were reacted at 65.degree. C. for 30 minutes. The absorbance at wavelength 570 nm was measured by a spectrophotometer and was compared to that of the control strain KCCM11016P.DELTA.aceE/pCR2.1-aceE (WT), and 256 modified strains showing an absorbance with at least 10% increase were selected. Other colonies showed similar or reduced absorbance compared to that of the control strain.

[0063] <Selective Medium (pH 8.0)>

[0064] Glucose 10 g, (NH.sub.4).sub.2SO.sub.4 5.5 g, MgSO.sub.4.7H.sub.2O 1.2 g, KH.sub.2PO.sub.4 (0.8 g, K.sub.2HPO.sub.4 16.4 g, Biotin 100 .mu.g, Thiamine HCl 1000 .mu.g, Calcium-Pantothenic Acid 2000 .mu.g, and Nicotinamide 2000 .mu.g (based on 1 L of distilled water)

[0065] The selected 256 strains were subjected to the ninhydrin reaction in the same manner as described above, and the top 53 kinds of strains with improved ability to produce L-lysine compared to that of KCCM11016P.DELTA.aceE/pCR2.1-aceE(WT) strain were selected.

Example 4: Confirmation of Abilities to Produce L-Lysine for Strains Selected from the E1p Artificial Variant Library

[0066] For comparison of the abilities to produce L-lysine for the 53 different strains selected in Example 3, the strains were cultured by the method described below and the components of the cultures obtained thereof were analyzed.

[0067] Each of the strains was inoculated into 250 mL corner-baffle flasks containing 25 mL of a seed culture medium, respectively, and cultured in a shaking incubator (200 rpm) at 30.degree. C. for 20 hours. Each of the 250 mL corner-baffle flasks containing 24 mL of an L-lysine producing culture was inoculated with 1 mL of a seed culture liquid, and cultured in a shaking incubator (200 rpm) at 30.degree. C. for 72 hours. The concentration of L-lysine in each culture was analyzed via HPLC.

[0068] <Seed Culture Medium (pH 7.0)>

[0069] Glucose (20 g), Peptone (10 g), Yeast Extract (5 g), Urea (1.5 g), KH.sub.2PO.sub.4 (4 g), K.sub.2HPO.sub.4 (8 g), MgSO.sub.4.7H.sub.2O (0.5 g), Biotin (100 .mu.g), Thiamine HCl (1000 .mu.g), Calcium-Pantothenic Acid (2000 .mu.g), Nicotinamide (2000 .mu.g) (based on 1 L of distilled water)

[0070] <Lysine-Producing Medium (pH 7.0)>

[0071] Glucose (100 g), (NH.sub.4).sub.2SO.sub.4 (40 g), Soybean Protein (2.5 g), Corn Steep Solids (5 g), Urea (3 g), KH.sub.2PO.sub.4 (1 g), MgSO.sub.4.7H.sub.2O (0.5 g), Biotin (100 .mu.g), Thiamine HCl (1000 .mu.g), Calcium-Pantothenic Acid (2000 .mu.g), Nicotinamide (3000 .mu.g), and CaCO.sub.3 (30 g) (based on 1 L of distilled water)

[0072] The top 10 strains with highest L-lysine concentrations were selected from the 53 different strains, and the culture and the analysis described above were performed repeatedly. The L-lysine concentrations analyzed are shown in Table 1 below.

TABLE-US-00003 TABLE 1 Concentration of L-Lysine Produced by 10 Selected Strains KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt) L-Lysine (g/L) Batch Batch Batch Strain 1 2 3 Mean Control KCCM11016P.DELTA.aceE/pCR2.1-aceE 42.1 41.9 41.7 41.9 (WT) 1 KCCM11016P.DELTA.aceE/pCR2.1-aceE 45.1 46.2 45.8 45.7 (mt)-1235 2 KCCM11016P.DELTA.aceE/pCR2.1-aceE 45.6 46.1 44.9 45.5 (mt)-1542 3 KCCM11016P.DELTA.aceE/pCR2.1-aceE 46.1 45.7 46.0 45.9 (mt)-3152 4 KCCM11016P.DELTA.aceE/pCR2.1-aceE 44.5 45.1 45.7 45.1 (mt)-5013 5 KCCM11016P.DELTA.aceE/pCR2.1-aceE 45.9 44.9 46.1 45.6 (mt)-5312 6 KCCM11016P.DELTA.aceE/pCR2.1-aceE 44.8 45.7 45.4 45.3 (mt)-6001 7 KCCM11016P.DELTA.aceE/pCR2.1-aceE 46.1 46.3 45.9 46.1 (mt)-7139 8 KCCM11016P.DELTA.aceE/pCR2.1-aceE 46.6 48.1 47.3 47.3 (mt)-8264 9 KCCM11016P.DELTA.aceE/pCR2.1-aceE 45.6 45.1 44.8 45.2 (mt)-9174 10 KCCM11016P.DELTA.aceE/pCR2.1-aceE 46.1 46.6 45.9 46.2 (mt)-9586

[0073] As a result of the analysis of L-lysine concentration, it was confirmed that the L-lysine yield of the 10 selected strains showed a maximum increase of 22% compared to the control strain, KCCM11016P.DELTA.aceE/pCR2.1-aceE (WT) strain.

Example 5: Confirmation of aceE Gene Modification of Strains Selected from the E1p Artificial Variant Library

[0074] In order to confirm the substitutions introduced in E1p of the 10 strains selected in Example 4, the polynucleotide sequences of modified aceE were analyzed. To determine the polynucleotide sequences, PCR was performed using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4).

TABLE-US-00004 primer 1 (SEQ ID NO: 3): 5'-TGGGA CCGGG AAACC GGG-3' primer 2 (SEQ ID NO: 4): 5'-GATTT ATCTG TCCCT TGA-3'

[0075] The polynucleotide sequences of each of the modified-type of aceE gene fragments obtained were confirmed by analysis, and compared with the polynucleotide sequence of SEQ ID NO: 2, and thus the amino acid sequences of the E1p variants were confirmed. The information on the substitutions of the E1p amino acid sequences of the selected strains is shown in Table 2 below.

TABLE-US-00005 TABLE 2 E1p Amino Acid Substitution of KCCM11016P/ pCR2.1-aceE (mt) of 10 Selected Strains E1p Amino Strain Acid Substitution KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-1235 Q432E KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-1542 E190V KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-3152 L438P KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-5013 Q195H KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-5312 P199S KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-6001 K435A KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-7139 Q432A KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-8264 Y418H KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-9174 N428A KCCM11016P.DELTA.aceE/pCR2.1-aceE (mt)-9586 Y201A

Example 6: Construction of a Vector for Introducing the E1p Variant on Chromosome

[0076] In order to confirm the application effect of the E1p variant confirmed in Example 5, a vector for introducing the same on the chromosome was constructed.

[0077] The primer 9 (SEQ ID NO: 12), which is introduced with an Xba I restriction site at 5' terminus, and the primer 10 (SEQ ID NO: 13), which is introduced with an Xba I restriction site at 3' terminus, were synthesized based on the reported polynucleotide sequences. The aceE (mt) gene fragments of 10 different modified-type of strains were amplified by PCR using the chromosomes of the selected 10 different strains as templates, respectively.

[0078] PCR was performed under the following conditions: denaturation at 94.degree. C. for 5 min; 30 cycles of denaturation at 94.degree. C. for 30 sec, annealing at 56.degree. C. for 30 sec, and polymerization at 72.degree. C. for 2 min; and polymerization at 72.degree. C. for 7 min.

TABLE-US-00006 primer 9 (SEQ ID NO: 12): 5'-AATCT AGATG GGACC GGGAA ACCGG G-3' primer 10 (SEQ ID NO: 13): 5'-AATCT AGAGA TTTAT CTGTC CCTTG A-3'

[0079] The PCR-amplified gene fragments of the 10 different strains were treated with Xba I to obtain the respective DNA fragments thereof, and the fragments were linked to the pDZ vector for introducing chromosomes including an Xba I restriction site, transformed into E. coli DH5.alpha., and streaked on solid LB medium containing kanamycin (25 mg/L).

[0080] The transformed colonies transformed with a vector inserted with a target gene were selected by PCR and the plasmids were obtained by the conventionally known plasmid extraction method. According to the modification inserted in E1p of each plasmid, the plasmids were named as pDZ-E1p (Q432E), pDZ-E1p (E190V), pDZ-E1p (L438P), pDZ-E1p (Q195H), pDZ-E1p (P199S), pDZ-E1p (K435A), pDZ-E1p (Q432A), pDZ-E1p (Y418H), pDZ-E1p (N428A), and pDZ-E1p(Y201A), respectively.

Example 7: Construction of a KCCM11016P-Derived Strain Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Lysine

[0081] Corynebacterium glutamicum KCCM11016P, an L-lysine producing strain, was transformed by a 2-step recombination of homologous chromosomes using the 10 vectors for introducing the Novel E1p variant prepared in Example 6. Then, the strains introduced with the E1p variant on the chromosomes were selected by analyzing the polynucleotide sequences, and the plasmids were named as KCCM11016P::E1p (Q432E), KCCM11016P::E1p (E190V), KCCM11016P::E1p (L438P), KCCM11016P::E1p (Q195H), KCCM11016P::E1p (P199S), KCCM11016P::E1p (K435A), KCCM11016P::E1p (Q432A), KCCM11016P::E1p (Y418H), KCCM11016P::E1p (N428A), and KCCM11016P::E1p (Y201A) according to the inserted E1p variant, respectively.

[0082] The strains were cultured in the same manner as in Example 4, and the L-lysine concentrations of the cultures were analyzed. For the measurement of the growth rate of the constructed strains, the residual glucose concentration of the cultures was measured 18 hours after the initiation of the culture (Table 3).

TABLE-US-00007 TABLE 3 Concentration (g/L) of Residual Glucose and L-Lysine Produced by KCCM11016P-Derived Strains Introduced with the E1p variant Batch 1 Batch 2 Batch 3 Mean residual residual residual residual Strain glucose L-lysine glucose L-lysine glucose L-lysine glucose L-lysine Control KCCM11016P 35.8 42.8 34.5 41.6 35.1 43.1 35.1 42.5 1 KCCM11016P::E1p 41.2 46.2 40.5 45.8 42.1 45.2 41.3 45.7 (Q432E) 2 KCCM11016P::E1p 43.2 45.2 41.8 45.8 43 46.7 42.7 45.9 (E190V) 3 KCCM11016P::E1p 40.5 45.1 41.1 45.9 40.6 45.8 40.7 45.6 (L438P) 4 KCCM11016P::E1p 38.1 44.5 37.5 45.2 38.5 44.9 38.0 44.9 (Q195H) 5 KCCM11016P::E1p 44.1 46.2 43.5 45.9 44.8 44.8 43.8 45.6 (P199S) 6 KCCM11016P::E1p 44.1 45.2 43.5 45.1 42.9 46.5 43.5 45.6 (K435A) 7 KCCM11016P::E1p 40.5 46.5 38.9 46.1 40.3 45.8 39.9 46.1 (Q432A) 8 KCCM11016P::E1p 38.5 47.5 37.6 46.6 39.1 46.5 38.4 46.9 (Y418H) 9 KCCM11016P::E1p 42.5 45.7 43.1 44.6 40.5 45.8 42.0 45.4 (Y201A) 10 KCCM11016P::E1p 39.8 46.5 40.1 47.9 40.3 46.2 40.1 46.9 (N428A)

[0083] The novel 10 different strains (i.e., KCCM11016P::E1p (Q432E), KCCM11016P::E1p (E190V), KCCM11016P::E1p (L438P), KCCM11016P::E1p (Q195H), KCCM11016P::E1p (P199S), KCCM11016P::E1p (K435A), KCCM11016P::E1p (Q432A), KCCM11016P::E1p (Y418H), KCCM11016P::E1p (Y201A), and KCCM11016P::E1p (N428A)), showed a slight decrease in glucose consumption rate while showing a maximum increase of 10% in lysine production, compared to those of the parent strain.

[0084] In this regard, the present inventors named KCCM11016P::E1p (N428A), the representative strain among the strains with improved ability to produce L-lysine, as Corynebacterium glutamicum "CA01-2289," and deposited the strain with the Korean Culture Center of Microorganisms, recognized as an international depositary authority under the Budapest Treaty, on Oct. 23, 2014, under the accession number KCCM11590P.

[0085] Upon examination, it was confirmed that the E1p variants of the 10 different strains (i.e., E1p (Q432E) (SEQ ID NO: 14), E1p (E190V) (SEQ ID NO: 15), E1p (L438P) (SEQ ID NO: 16), E1p (Q195H) (SEQ ID NO: 17), E1p (P199S) (SEQ ID NO: 18), E1p (K435A) (SEQ ID NO: 19), E1p (Q432A) (SEQ ID NO: 20), E1p (Y418H) (SEQ ID NO: 21), E1p (Y201A) (SEQ ID NO: 22), and E1p (N428A) (SEQ ID NO: 23)) were heavily distributed in two groups (amino acid residues at positions from 190 to 201, and amino acids at positions from 418 to 438).

[0086] Ten different strains, in which substitutions belonging to each group were included in combination, (i.e., (KCCM11016P::E1p (E190V, Q195H), KCCM11016P::E1p (E190V, P199S), KCCM11016P::E1p (Q195H, P199S), KCCM11016P::E1p (E190V, Y201A), KCCM11016P::E1p (Q195H, Y201A), KCCM11016P::E1p (P199S, Y201A), KCCM11016P::E1p (N428A, Q432E), KCCM11016P::E1p (N428A, K435A), KCCM11016P::E1p (Y418H, K435A), KCCM11016P::E1p (Y418H, Q432A)) were synthesized, and their abilities to produce L-lysine were measured in the same manner as described above (Table 4).

TABLE-US-00008 TABLE 4 Concentration (g/L) of Residual Glucose and L-Lysine Produced by KCCM11016P-Derived Strains Introduced with Combination of E1p substitutions Batch 1 Batch 2 Batch 3 Mean residual residual residual residual Strain glucose L-lysine glucose L-lysine glucose L-lysine glucose L-lysine Control KCCM11016P 35.2 42.1 34.6 42.5 35.6 42.5 35.1 42.4 1 KCCM11016P::E1p 47.1 47.1 46.5 48.1 46 43.1 46.5 46.1 (E190V, Q195H) 2 KCCM11016P::E1p 45.2 45.2 46.3 46.1 46 45 45.8 45.4 (E190V, P199S) 3 KCCM11016P::E1p 46.2 49.8 45.7 48.7 44 48.5 45.3 49.0 (Q195H, P199S) 4 KCCM11016P::E1p 42.8 46.1 43.1 45.7 42.5 46 42.8 45.9 (E190V, Y201A) 5 KCCM11016P::E1p 48.3 45.3 47.5 47.1 47 46.2 47.6 46.2 (Q195H, Y201A) 6 KCCM11016P::E1p 39.1 45.3 38.6 45.9 39.2 45.6 39.0 45.6 (P199S, Y201A) 7 KCCM11016P::E1p 45.9 48.1 47.1 49.2 46.5 49.3 46.5 48.9 (N428A, Q432E) 8 KCCM11016P::E1p 46.1 47.9 45.8 48.3 46.7 47.9 46.2 48.0 (N428A, K435A) 9 KCCM11016P::E1p 38.2 45.9 37.6 45.7 39.1 46 38.3 45.9 (Y418H, K435A) 10 KCCM11016P::E1p 41.2 46.1 40.9 45.1 39.9 47.2 40.7 46.1 (Y418H, Q432A)

[0087] As shown in Table above, all of the novel 10 strains with combined substitutions showed a slight decrease in glucose consumption rate while showing a maximum increase of 15.6% in the ability to produce L-lysine. The results confirm that a strain which is introduced with a combined substitution can exhibit improved ability to produce L-lysine than a strain which is introduced with single novel substitution.

[0088] The results confirm that the 10 strains with novel variants of E1p protein are effective strains which can significantly increasing an ability to produce L-lysine while not significantly reducing glucose consumption rate, compared to that of the parent strain, and also confirm that the amino acid residues at positions from 190 to 205, or from 415 to 440 are major regions that can exhibit the effects described above.

Example 8: Measurement of Activity of Pyruvate Dehydrogenase Complex (PDHC) Regarding Strains Introduced with the E1p Variant

[0089] The PDHC activity was measured regarding the selected strains by the method reported in the previous journal (Schreiner et al., J. Bacteriol. 187:6005, 2005). The control strains KCCM11016P and KCCM11016P.DELTA.aceE, and the 10 selected strains (KCCM11016P::E1p(Q432E), KCCM11016P::E1p(E190V), KCCM11016P::E1p(L438P), KCCM11016P::E1p(Q195H), KCCM11016P::E1p(P199S), KCCM11016P::E1p(K435A), KCCM11016P::E1p(Q432A), KCCM11016P::E1p(Y418H), KCCM11016P::E1p(Y201A), and KCCM11016P::E1p(N428A)), were inoculated into 25 mL of the seed culture medium specified in Example 4, and cultured until the late logarithmic phase.

[0090] The cells were collected by centrifugation and washed twice with 100 mM Tris-HCl (pH 7.2, 3 mM L-cysteine, 10 mM MgCl.sub.2) buffer solution, and finally suspended in 2 mL of the same buffer solution. The suspension of the cells was physically crushed by the general glass bead vortexing method for 10 minutes, and the supernatant was recovered by performing centrifugation (13,000 rpm, 4.degree. C., and 30 min) twice and used as a crude extract for the measurement of the activity of PDHC enzyme. For the measurement of the activity of PDHC enzyme, a reaction solution (10 mM MgCl.sub.2, 3 mM cysteine, 2 mM NAD, 0.9 mM thiamine diphosphate, 0.25 mM chlorpromazine, 6 mM pyruvate, 0.2 mM CoA in Tris-HCl buffer (pH 7.2)) for measuring the enzyme activity in an amount of 0.95 mL was added with 0.05 mL of the crude extract, and reacted at 30.degree. C. The activity unit of PDHC was defined in terms of NADH moles consumed per minute, and the measurement results of the enzyme activity are shown in Table 5 below.

TABLE-US-00009 TABLE 5 Measurement of Enzyme Activity of PDHC (%) Strain PDHC Activity (%) KCCM11016P 100 KCCM11016P.DELTA.aceE 0 KCCM11016P::E1p (Q432E) 39 KCCM11016P::E1p (E190V) 56 KCCM11016P::E1p (L438P) 53 KCCM11016P::E1p (Q195H) 55 KCCM11016P::E1p (P199S) 51 KCCM11016P::E1p (K435A) 53 KCCM11016P::E1p (Q432A) 42 KCCM11016P::E1p (Y418H) 36 KCCM11016P::E1p (Y201A) 46 KCCM11016P::E1p (N428A) 35

[0091] The PDHC activity of the strains introduced with novel variants showed the activity of 35% to 56% relative to the parent strain.

Example 9: Comparison of an Ability to Produce L-Lysine with aceE-Deletion Strain

[0092] For the comparative evaluation of KCCM11016P.DELTA.aceE, the aceE-deletion strain constructed in Example 2, and the 10 selected strains (KCCM11016P::E1p (Q432E), KCCM11016P::E1p (E190V), KCCM11016P::E1p (L438P), KCCM11016P::E1p (Q195H), KCCM11016P::E1p (P199S), KCCM11016P::E1p (K435A), KCCM11016P::E1p (Q432A), KCCM11016P::E1p (Y418H), KCCM11016P::E1p (Y201A), and KCCM11016P::E1p (N428A)), the strains were cultured in the same manner as in Example 4 using media containing ammonium acetate. The L-lysine concentration of the resulting cultures was analyzed by HPLC, and for the measurement of the growth rate of the constructed strains, the concentration of the residual glucose was measured 18 hours after the initiation of the culture (Table 6).

[0093] <Lysine-Producing Medium Containing Ammonium Acetate (pH 7.0)>

[0094] Glucose (100 g), CH.sub.3COONH.sub.3 (5 g), (NH.sub.4).sub.2SO.sub.4 (40 g), Soybean Protein (2.5 g), Corn Steep Solids (5 g), Urea (3 g), KH.sub.2PO.sub.4 (1 g), MgSO.sub.4.7H.sub.2O (0.5 g), Biotin (100 .mu.g), Thiamine HCl (1000 .mu.g), Calcium-Pantothenic Acid (2000 .mu.g), Nicotinamide (3000 .mu.g), and CaCO.sub.3 (30 g) (based on 1 L of distilled water)

TABLE-US-00010 TABLE 6 Concentration (g/L) of Residual Glucose and L-Lysine Produced by KCCM11016P-Derived Strains Introduced with E1p Variants and aceE-Deletion Strains Batch 1 Batch 2 Batch 3 Mean residual residual residual residual Strain glucose L-lysine glucose L-lysine glucose L-lysine glucose L-lysine Control KCCM11016P 45.7 40.9 45.6 42.1 46.7 41.3 46.0 41.4 Experimental KCCM11016P.DELTA.aceE 67.1 45.6 66.8 45.2 68.2 45.4 67.4 45.4 Group 1 KCCM11016P::E1p 48.2 43.7 46.5 44.2 47.5 43.4 47.4 43.8 (Q432E) 2 KCCM11016P::E1p 49.7 44.6 47.8 43.8 48.1 44.4 48.5 44.3 (E190V) 3 KCCM11016P::E1p 47.9 45.3 48.9 43.2 47.5 44.6 48.1 44.4 (L438P) 4 KCCM11016P::E1p 46.7 42.9 46.3 43.6 45.6 43.0 46.2 43.2 (Q195H) 5 KCCM11016P::E1p 50.4 45.2 50.6 45.1 51.2 44.6 50.7 45.0 (P199S) 6 KCCM11016P::E1p 51.3 44.9 50.9 45.2 52.7 45.0 51.6 45.0 (K435A) 7 KCCM11016P::E1p 48.9 45.6 47.8 46.0 47.9 45.9 48.2 45.8 (Q432A) 8 KCCM11016P::E1p 44.6 45.8 45.7 46.5 47.3 46.3 45.9 46.2 (Y418H) 9 KCCM11016P::E1p 47.6 43.8 46.7 44.5 48.7 43.7 47.7 44.0 (Y201A) 10 KCCM11016P::E1p 49.7 46.2 47.8 45.2 48.3 45.7 48.6 45.7 (N428A)

[0095] The KCCM11016P.DELTA.aceE strain showed an increase of 9.6% in the ability to produce L-lysine but a significantly reduced growth rate, compared to the parent strain. In contrast, the strains introduced with substitutions showed an increase in lysine yield and glucose consumption rate similar to when ammonium acetate was not added.

[0096] The results indicate that the strain introduced with the aceE variants can produce lysine in high yield without a noticeable effect in growth rate, compared to the aceE-deletion strain which can increase lysine yield with a significant decrease in growth rate.

Example 10: Construction of KFCC10750-Derived Strains Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Lysine

[0097] For confirming the effects of introduction of 10 novel variants in other strains of the genus Corynebacterium glutamicum, strains in which each of the 10 different E1p variants was respectively introduced into Corynebacterium glutamicum KFCC10750 (the above microorganism was first published as KFCC10750 and re-deposited to an international depositary authority under the Budapest Treaty and assigned the Accession No. KCCM11347P; Korean Patent No. 10-0073610), an L-lysine producing strain, were constructed in the same manner as in Example 7, and the strains were named as KFCC10750::E1p (Q432E), KFCC10750::E1p (E190V), KFCC10750::E1p (L438P), KFCC10750::E1p (Q195H), KFCC10750::E1p (P199S), KFCC10750::E1p (K435A), KFCC10750::E1p (Q432A), KFCC10750::E1p (Y418H), KFCC10750::E1p (Y201A), and KFCC10750::E1p (N428A). The eleven strains including the KFCC10750 strain as the control strain were cultured in the same manner as in Example 4, and the L-lysine concentration of the cultures of the strains was analyzed (Table 7).

TABLE-US-00011 TABLE 7 Concentration of L-Lysine Produced by KCCM10750P- Derived Strains Introduced with The E1p variant L-Lysine (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KFCC10750 38.8 38.1 37.9 38.3 1 KFCC10750::E1p (Q432E) 43.1 42.8 43.5 43.1 2 KFCC10750::E1p (E190V) 41.2 42.1 41.8 41.7 3 KFCC10750::E1p (L438P) 40.9 41.3 41.5 41.2 4 KFCC10750::E1p (Q195H) 41.6 42.3 41.8 41.9 5 KFCC10750::E1p (P199S) 42 42.4 41.9 42.1 6 KFCC10750::E1p (K435A) 41.8 42.1 42.2 42.0 7 KFCC10750::E1p (Q432A) 40.2 41.1 41.2 40.8 8 KFCC10750::E1p (Y418H) 44.5 44.9 44.8 44.7 9 KFCC10750::E1p (Y201A) 40.9 41 41.1 41.0 10 KFCC10750::E1p (N428A) 44.5 44.1 45.8 44.8

[0098] As a result, it was confirmed that the 10 different strains introduced with novel variants had a maximum increase of 17% of the ability to produce L-lysine compared to the parent strain.

Example 11: Construction of KCCM10770P-Derived Strains Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Lysine

[0099] For confirming the effects of introduction of 10 novel variants in other strains of the genus Corynebacterium glutamicum, strains in which each E1p variant was introduced into Corynebacterium glutamicum KCCM10770P (Korean Patent No. 10-0924065), an L-lysine producing strain, were constructed in the same manner as in Example 7, and the strains were named as KCCM10770P::E1p (Q432E), KCCM10770P::E1p (E190V), KCCM10770P::E1p (L438P), KCCM10770P::E1p (Q195H), KCCM10770P::E1p (P199S), KCCM10770P::E1p (K435A), KCCM10770P::E1p (Q432A), KCCM10770P::E1p (Y418H), KCCM10770P::E1p (Y201A), and KCCM10770P::E1p (N428A). The strains including the KFCC10750 strain as the control strain were cultured in the same manner as in Example 4, and the L-lysine concentration of the cultures of the strains was analyzed (Table 8).

TABLE-US-00012 TABLE 8 Concentration of L-Lysine Produced by KCCM10770P- Derived Strains Introduced with the E1p variant L-Lysine (g/L) Batch Strain 1 Batch 2 Batch 3 Mean Control KCCM10770P 48.1 47.5 47.8 47.8 1 KCCM10770P::E1p (Q432E) 53.3 53.4 54.1 53.6 2 KCCM10770P::E1p (E190V) 51.2 52.1 52.8 52.0 3 KCCM10770P::E1p (L438P) 51.5 51.3 50.7 51.2 4 KCCM10770P::E1p (Q195H) 51.9 52.1 52 52.0 5 KCCM10770P::E1p (P199S) 51.3 52.1 52.7 52.0 6 KCCM10770P::E1p (K435A) 52.4 52.6 51.4 52.1 7 KCCM10770P::E1p (Q432A) 50.2 51.2 50 50.5 8 KCCM10770P::E1p (Y418H) 55.8 54.5 54.1 54.8 9 KCCM10770P::E1p (Y201A) 49.9 50.3 50.7 50.3 10 KCCM10770P::E1p(N428A) 56.1 55.7 55.9 55.9

[0100] As a result, it was confirmed that the 10 strains introduced with novel variants had a maximum increase of 17% of the ability to produce L-lysine compared to the parent strain.

Example 12: Construction of CJ3P-Derived Strains Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Lysine

[0101] For confirming the effects of introduction of E1p variants in other strains of the genus Corynebacterium glutamicum, strains in which each E1p variant was introduced into Corynebacterium glutamicum CJ3P (Binder et al., Genome Biology 2012, 13: R40), an L-lysine producing strain, were constructed in the same manner as in Example 7, and the strains were named as CJ3P::E1p (Q432E), CJ3P::E1p (E190V), CJ3P::E1p (L438P), CJ3P::E1p (Q195H), CJ3P::E1p (P199S), CJ3P::E1p (K435A), CJ3P::E1p (Q432A), CJ3P::E1p (Y418H), CJ3P::E1p (Y201A), and CJ3P::E1p (N428A). The strains were cultured in the same manner as in Example 4, and the L-lysine concentration of the cultures of the strains was analyzed (Table 9).

TABLE-US-00013 TABLE 9 Concentration of L-Lysine Produced by CJ3P-Derived Strains Introduced with the E1p variant L-Lysine (g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control CJ3P 8.2 8.3 8 8.2 1 CJ3P::E1p (Q432E) 9.5 9.3 9.4 9.4 2 CJ3P::E1p (E190V) 8.8 9 8.7 8.8 3 CJ3P::E1p (L438P) 9.1 8.7 8.9 8.9 4 CJ3P::E1p (Q195H) 9.2 8.9 9 9.0 5 CJ3P::E1p (P199S) 9.3 9 9.1 9.1 6 CJ3P::E1p (K435A) 9.1 9 8.9 9.0 7 CJ3P::E1p (Q432A) 8.7 8.9 8.8 8.8 8 CJ3P::E1p (Y418H) 9.6 9.6 9.5 9.6 9 CJ3P::E1p (Y201A) 8.7 8.7 8.9 8.8 10 CJ3P::E1p (N428A) 9.7 9.8 9.8 9.8

[0102] As a result, it was confirmed that the 10 different strains introduced with novel variants had a maximum increase of 19.5% of the ability to produce L-lysine, compared to the parent strain.

[0103] The results indicate that each of the newly obtained 10 different strains with the E1p variant (E1p (Q432E), E1p (E190V), E1p (L438P), E1p (Q195H), E1p (P199S), E1p (K435A), E1p (Q432A), E1p (Y418H), E1p (Y201A), and E1p (N428A)) has excellent effect of increasing the ability to produce L-lysine, respectively.

Example 13: Construction of KCCM11201P-Derived Strains Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Valine

[0104] For confirming the effects of the 10 selected strains with E1p variants in other amino acids-producing strains of the genus Corynebacterium glutamicum, strains in which the E1p variant was introduced into Corynebacterium glutamicum KFCC11201P (Korean Patent No. 10-1117022), an L-valine producing strain, were constructed in the same manner as in Example 7, and the strains were named as KCCM11201P::E1p (Q432E), KCCM11201P::E1p (E190V), KCCM11201P::E1p (L438P), KCCM11201P::E1p (Q195H), KCCM11201P::E1p (P199S), KCCM11201P::E1p (K435A), KCCM11201P::E1p (Q432A), KCCM11201P::E1p (Y418H), KCCM11201P::E1p (Y201A), and KCCM11201P::E1p (N428A).

[0105] For the evaluation of the strains, each of the strains was inoculated into 250 mL corner-baffle flasks containing 25 mL of L-valine specified below, respectively, and cultured in a shaking incubator (200 rpm) at 30.degree. C. for 20 hours. The L-valine concentration in each culture was analyzed via HPLC (Table 10).

[0106] <Valine-Producing Medium (pH 7.2)>

[0107] Glucose (50 g), (NH.sub.4).sub.2SO.sub.4 (20 g), Corn Steep liquid (20 g), KH.sub.2PO.sub.4 (1 g), MgSO.sub.4.7H.sub.2O (0.5 g), Biotin (200 .mu.g), and CaCO.sub.3 (30 g) (based on 1 L of distilled water)

TABLE-US-00014 TABLE 10 Concentration of L-valine Produced by KCCM11201P-Derived Strains Introduced with the E1p variant L-valine (g/L) Strain Batch 1 Batch 2 Mean Control KCCM11201P 2.8 2.8 2.8 1 KCCM11201P::E1p (Q432E) 3.1 3.0 3.1 2 KCCM11201P::E1p (E190V) 3.3 3.2 3.3 3 KCCM11201P::E1p (L438P) 3.2 3.2 3.2 4 KCCM11201P::E1p (Q195H) 3.0 3.1 3.1 5 KCCM11201P::E1p (P199S) 3.1 3.3 3.2 6 KCCM11201P::E1p (K435A) 3.1 3.1 3.1 7 KCCM11201P::E1p (Q432A) 3.2 3.2 3.2 8 KCCM11201P::E1p (Y418H) 3.3 3.4 3.4 9 KCCM11201P::E1p (Y201A) 3.0 3.0 3.0 10 KCCM11201P::E1p (N428A) 3.4 3.4 3.4

[0108] As a result, it was confirmed that the 10 different strains introduced with novel variants had a maximum increase of 21% of the ability to produce L-valine, compared to the parent strain.

Example 14: Construction of Wild Type-Derived Strains Introduced with the E1p Variant and Comparison of their Abilities to Produce L-Valine

[0109] For reconfirming the ability to produce L-valine for the four different variants, among the 10 selected E1p variants, which showed the effect of a high increase in L-valine yield, strains in which each E1p variant was introduced into Corynebacterium glutamicum ATCC13032 were constructed in the same manner as in Example 7, and the strains were named as ATCC13032::E1p (E190V), ATCC13032::E1p (L438P), ATCC13032::E1p (Y418H), and ATCC13032::E1p (N428A).

[0110] For confirming the ability to produce L-valine for the above strains, each of the strains was transformed with pECCG117-DvalS (Korean Patent Application Publication No. 10-2014-0111421), which is an overexpression vector for L-valine biosynthesis, by electroporation. The transformed strains were obtained from selective media containing kanamycin (25 mg/L) and named as ATCC13032::E1p (E190V)_DvalS, ATCC13032::E1p (L438P)_DvalS, ATCC13032::E1p (Y418H)_DvalS, and ATCC13032::E1p (N428A)_DvalS.

[0111] The strains were cultured in the same manner as in Example 13 and the concentration of L-valine in each culture was analyzed (Table 11).

TABLE-US-00015 TABLE 11 Concentration of L-valine Produced by Wild Type-Derived Strains Introduced with the E1p variant L-valine (g/L) Strain Batch 1 Batch 2 Mean ATCC13032 0.1 0.1 0.1 Control ATCC13032_DvalS 1.3 1.2 1.3 1 ATCC13032::E1p (E190V) 0.1 0.1 0.1 2 ATCC13032::E1p (E190V)_DvalS 1.5 1.7 1.6 3 ATCC13032::E1p (L438P) 0.1 0.1 0.1 4 ATCC13032::E1p (L438P)_DvalS 1.4 1.4 1.4 5 ATCC13032::E1p (Y418H) 0.1 0.1 0.1 6 ATCC13032::E1p (Y418H)_DvalS 1.8 1.7 1.8 7 ATCC13032::E1p (N428A) 0.1 0.1 0.1 8 ATCC13032::E1p (N428A)_DvalS 1.9 1.7 1.8

[0112] As a result, it was confirmed that the four different strains introduced with novel variants had a maximum increase of 38% of the ability to produce L-valine, compared to the control strain.

[0113] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

Sequence CWU 1

1

331922PRTCorynebacterium glutamicum ATCC13032 1Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92022769DNACorynebacterium glutamicum ATCC13032 2atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360ccaggcatcg gcgtcggcgg acacatttcc acttacgcag gcgcagcccc tctgtacgaa 420gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg cattccgtcc 600taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840gttaactgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140tggaagcttc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260ggccacaact tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560gctaccacca tggcgactgt tcgtaccttc aaggaactga tgcgcgataa gggcttggct 1620gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800ggttccgtgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100ggcatcgacc gcatgtacgg cccaggcaag ggtgaagatg ttatctacta catcaccatc 2160tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340gttcgtgcca acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460cagctgaagc agacctccgg cccatacgtt gcagtgtctg acttctccac tgatctgcca 2520aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760gaggaataa 2769318DNAArtificial SequencePrimer 1 3tgggaccggg aaaccggg 18418DNAArtificial SequencePrimer 2 4gatttatctg tcccttga 18536DNAArtificial SequencePrimer 3 5gcaggtcgac tctagatgcg attcgcgtca aacgtg 36630DNAArtificial SequencePrimer 4 6gtcccttgag gtgatgtgaa tccatccact 30730DNAArtificial SequencePrimer 5 7agtggatgga ttcacatcac ctcaagggac 30836DNAArtificial SequencePrimer 6 8ccggggatcc tctagacgaa gcgccgtgag caattc 369521DNAArtificial SequenceaceE gene fragment comprising 5' end of aceE gene 9gcaggtcgac tctagatgcg attcgcgtca aacgtgagag aaacatcaca tctcgcggga 60aactacccga taattctttg caaaactttg caaagcggaa tgaacatgca gctagtttcc 120gtagaaatgt tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata 180cctgaaatcc cagtgagcgc accgctcccc ttacgtcaca gtctgtaaaa caaatcttcg 240gtgttgcgta tccttgttaa taacttatgc gttgactcat tcgtgcactt cggcgtgtca 300caattaggta cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat 360tccaacagga ggtgtggaaa tggccgatca agcaaaactt ggtggcaagc cctcggatga 420ctctaacttc gcgatgatcc gcgatggcgt ggcatcttat ttgaacgact cagatccgga 480ggagaccaac gagtggatgg attcacatca cctcaaggga c 52110538DNAArtificial SequenceaceE gene fragment comprising 3' end of aceE gene 10agtggatgga ttcacatcac ctcaagggac agataaatcc cgccgccaga cgttagtctg 60gcggcgggat tcgtcgtaaa gcaagctctt tttagccgag gaacgccttg tcagacaatg 120ttgcgccctt gatgttggcg aactcctgca gcaaatcgcg cacagtcaac ttcgacttgg 180tagcctgatc tgcctggtag acaatctggc cttcatgcat catgatcagg cgattgccca 240ggcgaattgc ctgttccatg ttgtgcgtga ccataagcgt agtcagattt ccatctgcca 300cgatcttttc ggtcaaggtg gtcacaagct ctgcacgctg tggatcaagc gctgcggtgt 360gctcatccaa cagcatgatt ttaggttgag taaaaccagc catcagcagg gacaatgcct 420gacgctgacc gccagagagc aaaccaactt tggcagtgag cctgttttcc agacccagct 480caaggcgctc aagttcctgc ttgaattgct cacggcgctt cgtctagagg atccccgg 538111029DNAArtificial SequenceDelta aceE 11gcaggtcgac tctagatgcg attcgcgtca aacgtgagag aaacatcaca tctcgcggga 60aactacccga taattctttg caaaactttg caaagcggaa tgaacatgca gctagtttcc 120gtagaaatgt tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata 180cctgaaatcc cagtgagcgc accgctcccc ttacgtcaca gtctgtaaaa caaatcttcg 240gtgttgcgta tccttgttaa taacttatgc gttgactcat tcgtgcactt cggcgtgtca 300caattaggta cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat 360tccaacagga ggtgtggaaa tggccgatca agcaaaactt ggtggcaagc cctcggatga 420ctctaacttc gcgatgatcc gcgatggcgt ggcatcttat ttgaacgact cagatccgga 480ggagaccaac gagtggatgg attcacatca cctcaaggga cagataaatc ccgccgccag 540acgttagtct ggcggcggga ttcgtcgtaa agcaagctct ttttagccga ggaacgcctt 600gtcagacaat gttgcgccct tgatgttggc gaactcctgc agcaaatcgc gcacagtcaa 660cttcgacttg gtagcctgat ctgcctggta gacaatctgg ccttcatgca tcatgatcag 720gcgattgccc aggcgaattg cctgttccat gttgtgcgtg accataagcg tagtcagatt 780tccatctgcc acgatctttt cggtcaaggt ggtcacaagc tctgcacgct gtggatcaag 840cgctgcggtg tgctcatcca acagcatgat tttaggttga gtaaaaccag ccatcagcag 900ggacaatgcc tgacgctgac cgccagagag caaaccaact ttggcagtga gcctgttttc 960cagacccagc tcaaggcgct caagttcctg cttgaattgc tcacggcgct tcgtctagag 1020gatccccgg 10291226DNAArtificial SequencePrimer 9 12aatctagatg ggaccgggaa accggg 261326DNAArtificial SequencePrimer 10 13aatctagaga tttatctgtc ccttga 2614922PRTArtificial SequenceE1p(Q432E) 14Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Glu 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn

Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92015922PRTArtificial SequenceE1p(E190V) 15Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92016922PRTArtificial SequenceE1p(L438P) 16Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Pro Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92017922PRTArtificial SequenceE1p(Q195H) 17Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu His Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro

Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92018922PRTArtificial SequenceE1p(P199S) 18Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92019922PRTArtificial SequenceE1p(K435A) 19Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875

880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92020922PRTArtificial SequenceE1p(Q432A) 20Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Ala 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92021922PRTArtificial SequenceE1p(Y418H) 21Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92022922PRTArtificial SequenceE1p(Y201A) 22Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565

570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92023922PRTArtificial SequenceE1p(N428A) 23Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92024922PRTArtificial SequenceE190V, Q195H 24Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser 180 185 190Arg Glu His Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92025922PRTArtificial SequenceE190V, P199S 25Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260

265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92026922PRTArtificial SequenceQ195H, P199S 26Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu His Gly Gly Ile Ser Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92027922PRTArtificial SequenceE190V, Y201A 27Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Val Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92028922PRTArtificial SequenceQ195H, Y201A 28Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu His Gly Gly Ile Pro Ser Ala Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92029922PRTArtificial SequenceP199S, Y201A 29Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Ser Ser Ala Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92030922PRTArtificial SequenceN428A, Q432E 30Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Glu 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro

Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92031922PRTArtificial SequenceN428A, K435A 31Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Ala Ala Thr His Gln 420 425 430Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92032922PRTArtificial SequenceY418H, K435A 32Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 92033922PRTArtificial SequenceY418H, Q432A 33Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser 180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys

Asn Leu Gln Arg Leu 275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415Gly His Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Ala 420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 920

Claims

1-14. (canceled)

15. A polypeptide having pyruvate dehydrogenase activity, wherein the polypeptide comprises an amino acid sequence having at least a 95% sequence identity to the SEQ ID NO: 1 and comprising at least one amino acid substitutions in a region of amino acids at positions from 190 to 205 of SEQ ID NO:1.

16. The polypeptide of claim 15, wherein the at least one amino acid substitution in the region of amino acids at positions from 190 to 205 of SEQ ID NO: 1 is selected from the group consisting of amino acids at positions 190, 195, 199, and 201

17. The polypeptide of claim 15, wherein the at least one amino acid substitution in the region of amino acids at positions from 190 to 205 of SEQ ID NO: 1 is selected from the group consisting of a substitution at position 190 from glutamic acid to valine (E190V), a substitution at position 195 from glutamine to histidine (Q195H), a substitution at position 199 from proline to serine (P199S), and a substitution at position 201 from tyrosine to alanine (Y201A).

18. The polypeptide of claim 15, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOS: 15, 17, 18, 22, and 24 to 29.

19. A polynucleotide encoding the pyruvate dehydrogenase variant of claim 15.

20. A vector comprising the polynucleotide of claim 19.

21. A microorganism of the genus Corynebacterium producing L-amino acid, wherein the microorganism comprises the pyruvate dehydrogenase variant of claim 15.

22. The microorganism of claim 21, wherein the microorganism is Corynebacterium glutamicum.

23. A method for producing L-amino acid, comprising: (a) culturing the microorganism of the genus Corynebacterium of claim 21 in a medium to produce an L-amino acid; and (b) recovering the L-amino acid from the cultured microorganism or the medium.